One of the biggest challenges for in situ heating transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) is the ability to measure the local temperature of the specimen accurately. Despite technological improvements in the construction of TEM/STEM heating holders, the problem of being able to measure the real sample temperature is still the subject of considerable discussion. In this study, we review the present literature on methodologies for temperature calibration. We analyze calibration methods that require the use of a thermometric material in addition to the specimen under study, as well as methods that can be performed directly on the specimen of interest without the need for a previous calibration. Finally, an overview of the most important characteristics of all the treated techniques, including temperature ranges and uncertainties, is provided in order to provide an accessory database to consult before an in situ TEM/STEM temperature calibration experiment.

The kinetics and mechanisms of interface reactions in a unidirectional continuous SiC fiber-reinforced Ti-17 matrix composite were investigated using transmission electron microscopy and scanning electron microscopy. It was found that a reaction zone (RZ) consisting of two-layered TiC-type carbide forms at the fiber/matrix interface during fabrication of the composite. After isothermal exposure at elevated temperatures, the two-layered TiC-type carbide is inherited, and a new TiC-type carbide layer forms within the RZ after exposure at temperatures lower than 900°C, while a new Ti3C2-type carbide layer forms after exposure at 900°C. It was also observed that the growth of RZ is a diffusion-controlled and temperature-dependent process, obeying the Fick's law-based parabolic relationship and the Arrhenius equation. Two material constants, the temperature-independent rate constant k0 and activation energy Q, are determined as 31.5 × 10−4µm/s1/2 and 49.9 kJ/mol, respectively.

A thin-walled copper (Cu)–tin (Sn) alloy cylinder was treated after spinning at 200–400°C for 0.5 h. The characteristics of the alloy microstructure under different temperatures were analyzed through electron back-scattered diffraction. The results were as follows. The grain size at 200–300°C decreases as the heat treatment temperature rises, but the grain size at 400°C increases. At 200–300°C, the microstructure primarily consists of deformed grains. It is found that the main reason for the formation of high-angle grain boundaries (HAGBs) is static recrystallization. For the grain boundary orientation differential, the low-angle sub-grain boundary gradually grows into the HAGB, and multiple annealing twin Σ9 boundaries appear. Grain orientation is generally random at any temperature range. The mechanical property test indicated that, at the upper critical recrystallization temperature of 300°C, the elongation of the Cu–Sn alloy gradually increases, and its yield strength and ultimate tensile strength rapidly decrease.

Atom probe tomography (APT) has emerged as an important tool in characterizing three-dimensional semiconductor devices. However, the complex structure and hybrid nature of a semiconductor device can pose serious challenges to the accurate measurement of dopants. In particular, local magnification and trajectory aberration observed when analyzing hybrid materials with different evaporation fields can cause severe distortions in reconstructed geometry and uncertainty in local chemistry measurement. To address these challenges, this study systematically investigates the effect of APT sampling directions on the measurement of n-type dopants P and As in an Si fin field-effect transistor (FinFET). We demonstrate that the APT samples made with their Z-axis perpendicular to the center axis of the fin are effective to minimize the negative effects that result from evaporation field differences between the Si fin and SiO2 on reconstruction and achieve improved measurement of dopant distributions. In addition, new insights have been gained regarding the distribution of ion-implanted P and As in the Si FinFET.

Taking advantage of recent advances in parallel computing, we studied compositional disorder along metal–oxygen atomic columns in a complex Mo,V-oxide bronze using multislice frozen-phonon calculations. Commonly, the virtual crystal approximation (VCA) is used to model compositional disorder at crystallographic sites in a unit cell for a number of different theoretical and experimental techniques. In the VCA, a weighted linear sum of atomic properties is used to approximate the model structure. When using the VCA, the extracted V content of Mo,V–O columns from experimental high-angle annular dark-field (HAADF) images will be about half the V content estimated from simulations, considering the distinct cation ordering. This discrepancy is larger than the spread of HAADF signals of different configurational orders at a given V concentration, which can be up to 20%. Certain “isophilic” atomic arrangements along the column can be distinguished from more random ones using HAADF-STEM imaging. The trends and ratios of the simulated intensity spreads due to different compositional ordering along 11 M–O columns along the c-axis of the Mo,V oxide bronze qualitatively match those observed in experimental HAADF-STEM data. Instrumental and sample-based noise adds to the variability but does not significantly distort the relative ratios of column intensity variation. We observed that we only required seven random configurations to represent the intensity variations along columns.

This study explores magnetization exhibited by nanoscale platinum-based structures embedded in pure silica plates. A superposition of laser pulses in the samples produced periodic linear arrangements of micro-sized structures. The samples were integrated by PtO2 microstructures (PtOΣs) with dispersed Pt oxide nanoparticles in their surroundings. The characterization of the materials was performed by high transmission electron microscopy studies. Furthermore, topographical and magnetic effects on the sample surfaces were analyzed by atomic force microscopy and magnetic force microscopy, respectively. The magnetic measurements indicated an enhancement in the gradient phase shift and in the gradient force related to the magnetic PtOΣs. The possibility of tuning the magnetic characteristics of the samples through contact with a Nd2Fe14B magnet was demonstrated. This process corresponds to an innovative method for obtaining magnetic PtOΣs induced by laser pulses. Moreover, an increase in the compactness of the silica with platinum-based structures was confirmed by an evaluation of the effective elastic modulus with reference to pure silica. The multimodal magnetic structures studied in this work seem to be candidates for developing high-density magnetic storage media.

When selecting a method for determining modal mineralogy and elemental composition of geological samples (e.g., meteorites), a number of factors should be considered, includingthe number of objects or the area to be analyzed; the scale of expected chemical variation; instrument time restrictions; and post-processing time. This study presents a method that minimizes acquisition time while maintaining the ability to distinguish minerals based on combinations of intensities of electron probe micro-analyzer-generated X-ray element maps. While some other methods yield similar outcomes, this method's post-processing utilizes standard parameterized, X-ray intensity “map math” in an algorithm that is adaptable and requires minimal supervision once implemented. This study's minimized supervision in the post-processing of X-ray intensity maps decreases analysis time and its adaptability increases the number of potential applications. The method also facilitates calibration of the exact locations of analysis using laser ablation methods. While the method described here has advantages, the choice of method always depends on the question being asked.

In this paper, an improved quantification technique for STEM/EDX measurements of 1D dopant profiles based on the Cliff-Lorimer equation is presented. The technique uses an iterative absorption correction procedure based on density models correlating the local mass density and composition of the specimen. Moreover, a calibration and error estimation procedure based on linear regression and error propagation is proposed in order to estimate the total measurement error in the dopant density. The proposed approach is applied to the measurement of the As profile in a nanodevice test structure. For the calibration, two crystalline Si specimens implanted with different As doses have been used, and the calibration of the Cliff-Lorimer coefficients has been carried out using Rutherford Back Scattering measurements. The As profile measurement has been carried out on an FinFET test structure, showing that quantitative results can be obtained in the nanometer scale and for dopant atomic densities lower than 1%. Using the proposed approach, the measurement error and detection limit for our experimental setup are calculated and the possibility to improve this limit by increasing the observation time is discussed.

Many nanoparticles in fields such as heterogeneous catalysis undergo surface structural fluctuations during chemical reactions, which may control functionality. These dynamic structural changes may be ideally investigated with time-resolved in situ electron microscopy. We have explored approaches for extracting quantitative information from large time-resolved image data sets with a low signal to noise recorded with a direct electron detector on an aberration-corrected transmission electron microscope. We focus on quantitatively characterizing beam-induced dynamic structural rearrangements taking place on the surface of CeO2 (ceria). A 2D Gaussian fitting procedure is employed to determine the position and occupancy of each atomic column in the nanoparticle with a temporal resolution of 2.5 ms and a spatial precision of 0.25 Å. Local rapid lattice expansions/contractions and atomic migration were revealed to occur on the (100) surface, whereas (111) surfaces were relatively stable throughout the experiment. The application of this methodology to other materials will provide new insights into the behavior of nanoparticle surface reconstructions that were previously inaccessible using other methods, which will have important consequences for the understanding of dynamic structure–property relationships.

In this work, the correlation between composition and relative evaporation field was investigated by tracking the statistics of multi-hit detector events in atom probe tomography (APT). This approach is applied systematically to a GaN-based nitride heterostructure with five AlxGa1−xN layers of varying Al composition. The relative field evaporation and the percentage of multi-hit events were found to increase with higher Al concentration. Furthermore, the comparison of the relative evaporation fields of AlN with respect to the constituent ions is found to be less than GaN with respect to its constituent ions. Despite equivalent compositions between opposing interfaces of the same AlxGa1−xN interlayer, the rate of change in multiplicity exhibits a consistent asymmetric trend with a steeper slope across the AlxGa1−xN/GaN interface compared to the GaN/AlxGa1−xN interface. The AlxGa1−xN/GaN heterostructure serves as a test structure for exploring field evaporation and neighborhood chemistry, which can be applied to any material chemistry and particularly other nitride systems.

Nanoporous, high-purity magnesium nitride (Mg3N2) was synthesized with a liquid ammonia-based process, for potential applications in optoelectronics, gas separation and catalysis, since these applications require high material purity and crystallinity, which has seldom been demonstrated in the past. One way to evaluate the degree of crystalline near-range order and atomic environment is electron energy-loss spectroscopy (EELS) in a transmission electron microscope. However, there are hardly any data on Mg3N2, which makes identification of electron energy-loss near-edge structure (ELNES) features difficult. Therefore, we have studied nanoporous Mg3N2 with EELS in detail in comparison to EELS spectra of bulk Mg3N2, which was analyzed as a reference material. The N-K and Mg-K edges of both materials are similar. Despite having the same crystal structure, however, there are differences in fine-structural features, such as shifts and absences of peaks in the N-K and Mg-K edges of nanoporous Mg3N2. These differences in ELNES are attributed to coordination changes in nanoporous Mg3N2 caused by the significantly smaller crystallite size of 2–6 nm compared to the larger (25–125 nm) crystal size in a bulk material.

Surfaces of polycrystalline ferritic Fe–Cr steel with grain sizes of about 13 µm in diameter were investigated with surface sensitive techniques. Thin oxide layers, with a maximum thickness of about 100 nm, were grown by oxidation in air at temperatures up to 450°C and were subsequently characterized using time-of-flight secondary ion mass spectrometry (TOF-SIMS) and atomic force microscopy. Correlative microscopy was applied, which allows for element-specific depth profiles on selected grains with a particular crystal orientation. A strong correlation between the grain orientation and the thickness of the oxide layer was found. The sequence in the oxidation growth rate of ferritic Fe–Cr steel crystal planes is found to be {011} > {111} > {001}, which is unexpectedly opposed to known Fe-based systems. Moreover, for the first time, the Cr/Fe ratio throughout the oxide layer has been determined per grain orientation. A clear order from high to low of {001} > {111} > {011} was detected.

A new transmission electron microscopy (TEM) specimen preparation method that utilizes a combination of focused ion beam (FIB) methods and ultramicrotomy is demonstrated. This combined method retains the benefit of site-specific sampling by FIB but eliminates ion beam-induced damage except at specimen edges and allows recovery of many consecutive sections. It is best applied to porous and/or fine-grained materials that are amenable to ultramicrotomy but are located in bulk samples that are not. The method is ideal for unique samples from which every specimen is precious, and we demonstrate its utility on fine-grained material from the one-of-a-kind Paris meteorite. Compared with a specimen prepared by conventional FIB methods, the final sections are uniformly thin and free from re-deposition and curtaining artifacts common in FIB specimens prepared from porous, heterogeneous samples.

Microchip technology with electron transparent membranes is a key component for in situ liquid transmission electron microscope (TEM) characterization. The membranes can significantly influence the TEM imaging spatial resolution, not only due to introducing additional material layers but also due to the associated bulging. The membrane bulging is largely defined by the membrane materials, thickness, and short dimension. The impact of the membrane on the spatial resolution, especially the extent of its bulging, was systematically investigated through the impact on the signal-to-noise ratio, chromatic aberration, and beam broadening. The optimization of the membrane parameters is the key component when designing the in situ TEM liquid cell. The optimal membrane thickness of 50 nm was found which balances the impact of membrane bulging and membrane thickness. Beyond this, the short membrane window dimension and the chip nominal spacing should be minimized. However, these two parameters have practical limitations in regards to chip handling.

The desire to image specimens in liquids has led to the development of open-cell and closed-cell techniques in transmission electron microscopy (TEM). The closed-cell approach is currently more common in TEM and has yielded new insights into a number of biological and materials processes in liquid environments. The open-cell approach, which requires an environmental TEM (ETEM), is technically challenging but may be advantageous in certain circumstances due to fewer restrictions on specimen and detector geometry. Here, we demonstrate a novel approach to open-cell liquid TEM, in which we use salt particles to facilitate the in situ formation of droplets of aqueous solution that envelope specimen particles coloaded with the salt. This is achieved by controlling sample temperature between 1 and 10°C and introducing water vapor to the ETEM chamber above the critical pressure for the formation of liquid water on the salt particles. Our use of in situ hydration enables specimens to be loaded into a microscope in a dry state using standard 3 mm TEM grids, allowing specimens to be prepared using trivial sample preparation techniques. Our future aim will be to combine this technique with an in situ light source to study photocorrosion in aqueous environments.

Heat-induced antigen retrieval (HIAR) treatment improves the antigen immunodetection in formalin-fixed, paraffin-embedded tissue samples and it can also improve the detection of intracellular antigens in alcohol-fixed cytological samples, although it could deleteriously impact immunodetection, particularly that of membranous antigens. We examined the differences in cell surface topography on MCF7 cells fixed in methanol/acetone (M/A) or 4% paraformaldehyde (4% PFA), as well as the changes caused by HIAR treatment at three different temperatures (60, 90, and 120°C), using atomic force microscopy. Furthermore, the consequences for immunostaining of five membranous antigens [epidermal growth factor receptor (EGFR), E-cadherin, CD9, CD24, and CD44] were examined. Our results illustrate that while there was no one single optimal immunostaining condition for the tested antibodies, the surface topography could be an important factor in successful staining. Generally, the best conditions for successful immunostaining were M/A fixation with no HIAR treatment, whereas in 4% PFA-fixed cells, HIAR treatment at 120°C was optimal. These conditions showed similarity in cell surface skewness. A correlation factor between successful immunocytochemical staining and the skewness parameter was 0.8000. Our results indicate that the presence of valleys, depressions, scratches, and pits on the cell surface is unfavorable for the successful immunodetection of cell surface antigens.

The seminiferous tubule (ST) is the location of spermatogenesis, where mature spermatozoa are produced with the assistance of Sertoli cells. The role of extracellular vesicles in the direct communication between Sertoli-germ cells in the ST is still not fully understood. In this study, we reported multivesicular bodies (MVBs) and their source of CD63-enriched exosomes by light and ultrastructure microscopy during the reproductive phases of turtles. Strong CD63 immunopositivity was detected at the basal region in the early and luminal regions of the ST during late spermatogenesis by immunohistochemistry (IHC), immunofluorescence (IF), and western blot (WB) analysis. Labeling of CD63 was detected in the Sertoli cell cytoplasmic processes that surround the developing germ cells during early spermatogenesis and in the lumen of the ST with elongated spermatids during late spermatogenesis. Furthermore, ultrastructure analysis confirmed the existence of numerous MVBs in the Sertoli cell prolongations that surround the round and primary spermatogonia during acrosome biogenesis and with the embedded heads of spermatids in the cytoplasm of Sertoli cells. Additionally, in spermatids, Chrysanthemum flower centers (CFCs) generated isolated membranes involved in MVBs and autophagosome formation, and their fusion to form amphiosomes was also observed. Additionally, autophagy inhibition by 3-methyladenine (after 24 h) increased CD63 protein signals during late spermatogenesis, as detected by IF and WB. Collectively, our study found MVBs and CD63 rich exosomes within the Sertoli cells and their response to autophagy inhibition in the ST during the spermatogenesis in the turtle.

The voltage-gated sodium channel [pore-forming subunit of the neuronal voltage-gated sodium channel (NaV1.6)] has recently been found in cardiac myocytes. Emerging studies indicate a role for NaV1.6 in ionic homeostasis as well as arrhythmogenesis. Little is known about the spatial organization of these channels in cardiac muscle, mainly due to the lack of high-fidelity antibodies. Therefore, we developed and rigorously validated a novel rabbit polyclonal NaV1.6 antibody and undertook super-resolution microscopy studies of NaV1.6 localization in cardiac muscle. We developed and validated a novel rabbit polyclonal antibody against a C-terminal epitope on the neuronal sodium channel 1.6 (NaV1.6). Raw sera showed high affinity in immuno-fluorescence studies, which was improved with affinity purification. The antibody was rigorously validated for specificity via multiple approaches. Lastly, we used this antibody in proximity ligation assay (PLA) and super-resolution STochastic Optical Reconstruction Microscopy (STORM) studies, which revealed enrichment of NaV1.6 in close proximity to ryanodine receptor (RyR2), a key calcium (Ca2+) cycling protein, in cardiac myocytes. In summary, our novel NaV1.6 antibody demonstrates high degrees of specificity and fidelity in multiple preparations. It enabled multimodal microscopic studies and revealed that over half of the NaV1.6 channels in cardiac myocytes are located within 100 nm of ryanodine receptor Ca2+ release channels.

Traumatic brain injury (TBI) is a main cause of death and disabilities in young adults. Although learning and memory impairments are a major clinical manifestation of TBI, the consequences of TBI on the hippocampus are still not well understood. In particular, how lesions to the sensorimotor cortex damage the hippocampus, to which it is not directly connected, is still elusive. Here, we study the effects of sensorimotor cortex ablation (SCA) on the hippocampal dentate gyrus, by applying a highly sensitive gray-level co-occurrence matrix (GLCM) analysis. Using GLCM analysis of granule neurons, we discovered, in our TBI paradigm, subtle changes in granule cell (GC) morphology, including textual uniformity, contrast, and variance, which is not detected by conventional microscopy. We conclude that sensorimotor cortex trauma leads to specific changes in the hippocampus that advance our understanding of the cellular underpinnings of cognitive impairments in TBI. Moreover, we identified GLCM analysis as a highly sensitive method to detect subtle changes in the GC layers that is expected to significantly improve further studies investigating the impact of TBI on hippocampal neuropathology.